Hydrogel is the most common and important substance with high water content but insoluble in water. It can absorb water in amounts up to several hundredfold over its own weight while still keeping its shape. Natural gels existing inside of most organisms and plants, and many chemically synthesized gels belong to hydrogels. Macromolecular gels are a kind of common hydrogel. They have a three-dimensional crosslinked network structure composed of the main chains of macromolecules and also side chains containing hydrophilic (polar) groups, hydrophobic groups and/or dissociable groups with the solvent entrapped in the molecular network. The crosslinked sites of the macromolecular gel network can be either formed by chemical-crosslinking through covalent bonds, or formed by physical-crosslinking through electrostatic interaction, hydrogen-bond interaction, hydrophobic interaction, etc.
Hydrogels, especially the macromolecular hydrogels prepared with the extracellular matrix materials, are widely used in the field of biomedicine. Compared with the hydrogels prepared with synthetic materials, the hydrogels prepared with the extracellular matrix have many advantages, such as simulating the natural environment as the inside of organisms, very high water content, good permeability, better biocompatibility, and adjustable enzymatic degradability. More importantly, the extracellular matrix may possess bioinduction functions, can direct and induce specific regeneration of tissues. For example, sodium hyaluronate is a natural extracellular matrix macromolecule, possessing biological functions such as managing cell adhesion and migration, and regulating cell division and differentiation. The high molecular weight sodium hyaluronate can induce a chicken embryonic limb marrow stem cell to be differentiated into a cartilage cell. Therefore, in the biomedicine (particularly the tissue engineering) field more and more attention is focused on the hydrogel prepared with extracellular matrix.
Although hydrogels have a number of advantages, the method of administration significantly limits its wide application in the biomedicine field. Currently many medical products of hydrogels are formulated into films and porous sponges etc., e.g. gelatin sponge and collagen sponge. Usually these products can only be used topically or in laparotomy. However, with the development of the medical technology, more and more doctors and patients are inclined to the minimally invasive surgery, requesting that the medical products can also be used under endoscopes, which present a new challenge for developing medical products.
The injectable hydrogel medical products can be used either under an endoscope or in combination with a minimally invasive surgery. They are also suitable for three-dimensional wounds of any complicated shape, and can adhere to the wounds very well, having prospects of wide application in the biomedicine field. For example, currently as the new-generation anti-wrinkle fillers, various injectable crosslinked hydrogels made from sodium hyaluronate that overcome the immunogenicity risk of the collagen anti-wrinkle filler, have been widely used in cosmetology. As representative products of such new-generation anti-wrinkle fillers, Restylane (Q-Med, Sweden), Hylaform (Inamed Corporation, the United States), Juvederm (Leaderm, France), Belotero (Anteis, Switzerland), and Puragen (Mentor Corporation, the United States) are commercially available in Europe (among them Restylane and Hylaforms have further been approved by the FDA of the United States).
Currently, most medical products of hydrogels are prepared by chemical crosslinking followed by purification to remove residual crosslinking agents and byproducts. However, the chemical crosslinking agents usually have great toxic and side effects, and even a complicated process can hardly guarantee to remove them completely. More seriously, the residual crosslinking agents with part of functional groups crosslinked, have been immobilized in the hydrogel via covalent bonds, and cannot be removed. These residual crosslinking agents may cause toxicity and side effects such as inflammation in clinicals. For example, trace amounts of residual crosslinking agents in the gelatin sponge may cause a serious inflammation response in organisms. The previously described injectable crosslinked hydrogels made from sodium hyaluronate are also prepared by the process of crosslinking first and then purification. For example, Restylane and Hylaforms have been prepared by chemical reactions between the hydroxyl group of sodium hyaluronate and 1,4-tetramethylene glycol diglycidyl ether or divinyl sulfoxide. However, the residual 1,4-tetramethylene glycol diglycidyl ether or divinyl sulfoxide are very difficult to remove completely from the hydrogel, and those with one function group being reacted and fixed in the hydrogel via the covalent bond cannot be removed. This limitation not only requires a complicated purifying process, but also raises clinical risks.
Recently, disulfide-bond crosslinked hydrogels have been investigated. This disulfide bond is a reversible chemical bond, free thiol groups can be oxidized into disulfide bonds which can be reduced back into free thiol groups. For example, currently the disulfide-bond crosslinked hydrogel has been used as a cell culture matrix, and the cells can be recovered very conveniently by adding cell-compatible reducer of disulfide bonds.
Oxidants (i.e. hydrogen peroxide, iodine, alkyl peroxide, acid peroxide, dimethyl sulfoxide, Fe3+, Co3+, Ce4+, etc.) can oxidize thiol groups into disulfide bonds. However, these oxidants usually have certain toxic and side effects, and are highly harmful if left in the medical products; moreover their oxidation capacity is too strong, and the reaction is so vigorous that the disulfide bond will be further oxidize into byproducts such as sulfonate.
Oxygen can also oxidize free thiol groups into disulfide bonds. One oxygen gas molecule can oxidize four thiol groups into two disulfide bonds, and produce two water molecules as well, without any other byproducts. Preparing disulfide-bond crosslinked hydrogels with oxygen gas as the oxidant has many advantages, such as simple and mild reaction conditions and no need for a crosslinking agent. By using oxygen gas as the crosslinking agent to prepare disulfide-bond crosslinked hydrogels, it is hopeful to break the limitation of the residual crosslinking agent in the hydrogel preparation process as described above.
Disulfide-bond crosslinked hydrogels have many potential applications in the biomedicine field, and have been paid much attention in recent years. So far, however, there has been no report about their practical clinical applications, and two major reasons are responsible for that. The first is that the current preparation process of the disulfide-bond crosslinked hydrogel is not suitable for industrialized production. Using oxygen gas to oxidize the thiol group into the disulfide-bond crosslinked hydrogel under physiological conditions is a slow process, which needs to continuously consume a lot of oxygen. It is widely accepted by those skilled in the art that making the solution open to air is a precondition for forming the disulfide-bond crosslinked gel. In the current disclosed reports, all the biocompatible macromolecule solutions containing thiol groups need to be open to air for forming the disulfide-bond crosslinked gel. For example, thiolated sodium hyaluronate derivative solution can form the disulfide-bond crosslinked gel when being open to air, and produce a disulfide-bond crosslinked film after being dried; the mixed solution of thiolated sodium hyaluronate derivative and thiolated collagen derivative can form the disulfide-bond crosslinked gel when being open to air, and produce the disulfide-bond crosslinked film or porous sponge after being dried at the normal or freezing temperatures. The second for preventing the disulfide-bond crosslinked hydrogel from being used in the clinical practice is the product form. Most of the disulfide-bond crosslinked gels currently reported are prepared in the form of film or sponge, and can only be used topically or in laparotomy, not meeting the requirements of many clinical therapies (especially the minimally invasive surgery).
So far there has been a widespread technical prejudice among those skilled in the art: The biocompatible macromolecule solution containing the thiol group needs to be open to air for forming the disulfide-bond crosslinked hydrogel. To a great extent, this prejudice limits the large-scale industrialized production process of the disulfide-bond crosslinked hydrogel. So far there has been no report about preparing the disulfide-bond crosslinked hydrogel in a sealed injectable container, although this injectable disulfide-bond crosslinked hydrogel has very wide application in the biomedicine field.